Research will continue on pyridoxal kinase and pyridoxine-5-P oxidase, as well as 4-aminobutyrate aminotransferase with special emphasis on the following aspects: a) Crosslinking of pyridoxal kinase and pyridoxine-5-P oxidase. Research in our laboratory indicates that bis-pyridoxal-5-P can be used as a crosslinker of subunits of enzymes that form macromolecular complexes. Since the kinase remains fully active after preincubation with bis-pyridoxal-5-P followed by reduction with NaBH4, we are going to attempt the crosslinkage of the kinase with the apoprotein of the oxidase. The crosslinked species will be separated by gel filtration chromatography, DEAE-Stephadex chromatography and isoelectric focusing. The technique of SDS polyacrylamide gel electrophoresis will be used to detect the formation of macromolecular species of molecular weight greater than 60,000. After separation of the crosslinked species, one should be able to study their catalytic activities and the effect of crosslinking on the transient time. b) The use of fluorescence substrates as probes of the catalytic site of pyridoxine-5-P oxidase. New synthetic substrates prepared in our laboratory, P-pyridoxyl-m-aminobenzoate and P-pyridoxyl-P-aminobenzoate, will be used as probes of the microenvironment of the catalytic site of pyridoxine-5-P oxidase. The spectroscopic properties of the synthetic substrates, absorption band centered at around 320 nm, emission band at around 415 nm, and a suitable fluorescence decay (6.5 nanoseconds) can be used conveniantly to explore the physical properties of the substrate binding site. c) Further characterization of the non-equivalent binding sites of 4-aminobutyrate aminotransferase.